As a method for efficiently producing nucleoside-5′-phosphates at a low cost by biochemically phosphorylating nucleosides, there was developed a method of efficiently producing a nucleoside-5′-phosphate without generation of byproducts of nucleoside-2′-phosphate acid and nucleoside-3′-phosphate isomers, which comprises allowing cells of a particular microorganism to act on a nucleoside and a phosphate donor selected from the group consisting of polyphosphoric acid (salt thereof), phenyl phosphate (salt thereof) and carbamyl phosphate under an acidic condition (Japanese Patent Laid-open (Kokai) No. 7-231793/1995).
Then, it was confirmed that the productivity of nucleoside-5′-phosphates can further be improved by obtaining a gene coding for an acid phosphatase from Escherichia blattae or Morganella morganii and expressing the gene in Escherichia coli in a large scale using genetic engineering techniques.
The structure of the acid phosphatase is shown in FIG. 2. That is, FIG. 2 shows alignment of the amino acid sequence of the acid phosphatase derived from Escherichia blattae (abbreviated as “EB-AP” hereinafter) with the amino acid sequences of acid phosphatases derived from Morganella morganii, Salmonella typhimurium and Zymomonas mobilis. The asterisks indicate conserved residues. The regions of the secondary structure are indicated with bars over the aligned sequences. The boxed portions are motives commonly observed in the acid phosphatase family. The motives consist of three domains, 1) KXXXXXXRP (SEQ ID NO: 121), 2) PSGH (SEQ ID NO: 122) and 3) SRXXXXXHXXXD (SEQ ID NO: 123). In these motives, X represents an arbitrary amino acid residue.
Although these acid phosphatases (FIG. 2) have transphosphorylation activity, they suffer from a drawback that their phosphatase activity for decomposing nucleoside-5′-phosphate into a nucleoside is dominant in wild-type strains, and hence accumulated nucleoside-5′-phosphate will be decomposed. Therefore, a large number of mutant enzymes were generated by the random mutagenesis method, and a mutant acid phosphatase having relatively improved transphosphorylation activity compared with phosphatase activity was found among the mutant enzymes. And it was demonstrated that the productivity of nucleoside-5′-phosphate was drastically improved by abundantly expressing a gene coding for the mutant acid phosphatase gene (Japanese Patent Laid-open (Kokai) No. 8-535568/1996).
This mutant acid phosphatase has improved affinity for a nucleoside, and it is thought that the transphosphorylation activity was improved by the enhanced affinity.
It was demonstrated that the aforementioned mutant acid phosphatase derived from Escherichia blattae (=G74D/I153T mutant enzyme (mutant enzyme in which 74th Gly is replaced with Asp and 153rd Ile is replaced with Thr, the mutation pattern will be similarly represented hereinafter)) showed weaker transphosphorylation activity compared with a corresponding G72D/I151T mutant enzyme of acid phosphatase derived from Morganella morganii (MM-AP), whereas a 10-residue replaced L63Q/A65Q/E66A/N69D/S71A/S72A/G74D/T135K/E136D/I153T mutant enzyme (referred to simply as “10-residue replaced mutant EB-AP” hereinafter), in which 8 amino acid residues were replaced with the amino acids of MM-AP that correspond on the primary structure basis, showed transphosphorylation activity substantially comparable to that of G72D/I151T mutant MM-AP (Japanese Patent Laid-open (Kokai) No. 9-161674/1997).
A method for producing a nucleoside-5′-phosphate has been established by expressing a large amount of the aforementioned G74D/I153T mutant enzyme gene, or the 10-residue replaced mutant enzyme gene in Escherichia coli (Japanese Patent Laid-open (Kokai) Nos. 9-37785/1997 and 10-201481/1999). However, a mutant EB-AP with further improved productivity is still desired.